Article pubs.acs.org/jmc
Distinct Temporal Fingerprint for Cyclic Adenosine Monophosphate (cAMP) Signaling of Indole-2-carboxamides as Allosteric Modulators of the Cannabinoid Receptors Erin E. Cawston,† Mark Connor,‡ Vincenzo Di Marzo,§ Romano Silvestri,∥ and Michelle Glass*,† †
Centre for Brain Research and Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand ‡ Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, 2 Technology Place, Sydney, NSW 2109, Australia § Istituto di Chemica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, Comprensorio Olivetti, I-80078 Pozzuoli, Napoli, Italy ∥ Istituto Pasteur − Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185, Rome, Italy ABSTRACT: ORG27569 (1) is an allosteric modulator of CB1. 1 produces a distinct cAMP temporal fingerprint with complex time-dependent modulation of agonist-mediated responses. The aim of this study was to characterize the cAMP signaling response of indole-2-carboxamides structurally correlated to 1 for both CB1 and CB2. We show that at CB1 1, 10, 13, and 18 display a delay in inhibiting CP55,940-mediated cAMP inhibition, whereas compounds 7, 14, 15, 16, 20, and 22 act immediately. To further characterize this, compounds 1, 10, 13, 14, 15, 18, and 20 were tested for their influence on CP55,940mediated hyperpolarization in AtT20-hCB1 cells. Intriguingly, all compounds generated a response similar to that of 1, producing no decrease in CB1-mediated peak hyperpolarization at concentrations up to 10 μM but enhancing the rate at which the channel repolarizes. Additionally, we show that compounds 5, 10, and 20 indole-2-carboxamides modulate cAMP signaling through CB2.
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INTRODUCTION The endocannabinoid system principally comprises cannabinoid receptors 1 and 2 (CB1 and CB2, respectively) along with the endogenous cannabinoid receptor ligands.1 CB1 is present at high levels throughout the central nervous system2 and acts as a modulator of neurotransmitter release. CB1 is of considerable therapeutic interest for the treatment of neurodegenerative disease,3,4 pain,5 and multiple sclerosis.6,7 CB2 is mainly expressed in peripheral tissues, particularly immune cells8,9 as well as in CNS microglia10 and therefore is a therapeutic target for the treatment of pain and inflammatory conditions. Biased ligands and allosteric modulators provide an opportunity for improved cannabinoid based drug therapies. CB1 has been shown to have an allosteric binding site,11 whereas no allosteric site has been described to date for CB2. Several CB1 allosteric modulators have been described, with Price et al.11 reporting a series of structurally related small molecules, the most potent being 5-chloro-3-ethyl-N-(4(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (1, ORG27569), which was shown to be an allosteric enhancer © XXXX American Chemical Society
of the (−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940) binding but produced insurmountable antagonism (allosteric inhibition) in GTPγS binding assays. Subsequent studies have suggested that 1 is a biased ligand activating β-arrestin-1mediated signaling, resulting in the phosphorylation of pERK. However, there are conflicting results on the extent of this signal and whether this is G-protein-mediated.12−14 Horswill et al.13 subsequently described 1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea (4, PSNCBAM-1) as an allosteric modulator, which also enhanced orthosteric agonist binding while antagonising GTPγS activation and the ability of CB1 to reduce mIPSC frequency in cerebellar neurons.15,16 In our previous study, we suggested that 1 and 4 have a distinct temporal cAMP fingerprint, with complex, concentration and time-dependent modulation of agonist-mediated regulation of cAMP levels.17 Thus, at submaximal concentrations of 1 a marked delay in the ability to antagonize agonistReceived: April 13, 2015
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DOI: 10.1021/acs.jmedchem.5b00579 J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Article
Table 1. Initial cAMP Screen Data for Indole-2-carboxamide Compounds and Reference Compound 1a
a N: difference not statistically significant from controls. Y: difference statistically significant from controls. One phase association curves were fitted for individual data sets, and the plateau of the curves was obtained from three independent experiments in the presence or absence of EC90 CP55,940 ́ k post-test to assess multiple comparisons in parametric ANOVA (CP) with 5 μM forskolin. Statistical significance was determined using Holm-Šidá RM tests. Statistically significant p-values are shaded in gray.
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RESULTS AND DISCUSSION Initial Pharmacological Evaluation of Compounds via Functional cAMP Signaling. Determination of the impact of compound 1 and 14 indole-2-carboxamide derivatives (Table 1) on cannabinoid receptor cAMP signaling was carried out using a kinetic cAMP assay.19 This real-time kinetic BRET CAMYEL assay has previously been used to characterize the cAMP signaling of 1 and 4.17 In this previous study, we showed that the action of these compounds was not due to noncompetitive antagonism of signaling but that instead enhanced binding results in an increased rate of receptor desensitization and delayed internalization which results in time-dependent modulation of cAMP signaling. Our previous study showed that HEK 3HA-hCB1 cells produced a rapid increase in cAMP in response to forskolin that
mediated cAMP inhibition was observed, followed by inverse agonism. This was suggested to be mediated by an increased rate of desensitization of the receptor in the presence of 1. In this study, we have characterized the temporal nature of allosteric antagonism of cAMP signaling for a previously described series of 1-derived indole-2-carboxamide compounds.18
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CHEMISTRY
All indole-2-carboxamides used in this work were synthesized and described by Piscitelli and colleagues in 2012.18 Compound numbering has been kept the same as in the original description18 to allow for ease of comparison. B
DOI: 10.1021/acs.jmedchem.5b00579 J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Article
plateaued within approximately 5 min and was maintained for the entire time course of the assay (approximately 30 min).17 We have previously shown that the HEK-hCB1 forskolinmediated response could be inhibited with the orthosteric agonist CP55,940 with an EC50 = 0.437 nM ± 0.136 nM.17 HEK-hCB2 was characterized for its response to CP55,940 demonstrating inhibition of forskolin stimulated cAMP with an EC50 = 0.576 nM ± 0.168 nM. Compound 1 and the 14 derived indole-2-carboxamide compounds (Table 1) were screened for their ability to modulate the inhibition of forskolin (5 μM) stimulated cAMP by approximate EC90 levels of CP55,940 (5 nM) to test for allosteric modulation of the orthosteric response or alone at a single high concentration (10 μM) to test for direct allosteric agonism/inverse agonism. Data were assessed by fitting one phase association curves and identifying if the plateau of the curve was statistically different from forskolin (in the absence of CP55,940, Figure 1C) or forskolin plus CP55,940 (Figure 1B). Table 1 describes the results of the initial cAMP screening. Figure 1A shows the N-phenylethyl-1H-indole-2-carboxamide scaffold in reference to Table 1. Figure 1B illustrates representative data from 7 compounds tested in the initial screen against CB1 and the fitting of the one phase association curves for assessments of statistical significance. This figure shows the forskolin-mediated response which is inhibited by EC90 of CP55,940. From the initial screen of CB1 shown in Table 1, compounds 1, 7, 10, 13, 14, 15, 16, 18, 20, and 22 all resulted in statistically significantly different cAMP curves in the presence of EC90 CP55,940. Additionally, in the absence of orthosteric ligand, compounds 1, 10, 11, 13, 18, and 20 produced a stimulation of cAMP above that produced by forskolin alone. As shown in Table 1, four compounds were identified to produce alterations in cAMP levels statistically different from controls with regard to the CB2 receptor either in the presence (compounds 10 and 11) or absence (compounds 5, 11, and 20) of the orthosteric ligand. Those compounds that produced significant effects at 10 μM were then further characterized as described below. All compounds identified for further characterization were first tested with HEK-WT cells to ensure that any cAMP signaling detected was either CB1- or CB2-mediated. Compound 11 was identified as causing a significant decrease in cAMP (P =
